PROJECT SUMMARY The hallmarks of the adaptive immune system are diversity and memory. A diverse T cell repertoire is critical to ensure an effective defense against a near infinite number of potential viral, fungal, and bacterial pathogens. T cell diversity is generated in the thymus. Due to imprecise T cell receptor assembly, over 108 T cell clones are produced in the thymus, each expressing a unique T cell receptor. During early adulthood, the thymus involutes. Therefore, to maintain a healthy immune system, T cell diversity has to be sustained in the immunological periphery. Maintenance of T cell diversity requires fine tuning because small perturbations in the T cell pool can have dramatic effects over time on T cell diversity. Each clone must be supported by positive homeostatic signals that enable survival over decades. At the same time, each clone must not overproliferate. Because the immunological space appears to be fixed, overexpansion of individual T cell clones can potentially crowd out its neighbors. Despite the fundamental importance of T cell homeostasis to human health and disease, we lack mechanistic insight into how T cell diversity is maintained. There are two important barriers to progress. For one, human studies have often focused on relatively small, unrelated cohorts of phenotypically heterogeneous aged individuals. Secondly, the study of a complex heterogeneous cell population requires assays with single cell resolution. To overcome these barriers, we propose to study a phenotypically similar cohort of human outliers who have profound alterations in their T cell repertoire, namely patients with cutaneous T cell lymphoma (CTCL). CTCL is an incurable non-Hodgkin lymphoma of the skin- homing CD4+ T cell. Patients with advanced leukemic CTCL invariably develop overproliferation of the malignant T cell clone and profound losses of the benign, untransformed CD4+ and CD8+ T cells. This lymphopenia is a characteristic feature of disease, develops in a stage-dependent manner, and is inevitable in patients with Stage IV disease. Presumably, the loss of normal T cells benefits the tumor cells by inhibiting anti-tumor immunity. Emerging data suggest that these two antiparallel trends occur due to pathological alterations in the homeostatic signals that keep clones alive and the homeostatic signals that keep clones from overcrowding the population. We hypothesize that the CTCL tumor cells have hyperactivation of the positive homeostatic signals and resistance to the negative homeostatic signals. Conversely, the disappearing, benign T cells in CTCL patients are relatively resistant to the positive homeostatic signals and highly sensitive to the negative homeostatic signals produced by CTCL cells. To elucidate disease mechanisms, we will use high- dimension discovery based approaches to identify and functionally validate high priority molecules that govern T cell homeostasis. We believe that successful completion of this proposal will identify important homeostatic signaling mechanisms that are fundamentally important for T cell homeostasis in health and in other diseases.